The present invention relates generally to the diagnosis of cancer and specifically to identification of a hypermethylated glutathione-S-transferase (GSTP1) gene as a diagnostic indicator of hepatic cell proliferative disorders.
Hepatocellular carcinoma (HCC) constitutes one of the most common life-threatening cancers in world. Most HCC cases arise in the setting of chronic hepatitis virus infection. Dietary carcinogens, such as alflatoxin B1, likely also contribute to hepatic carcinogenesis. Glutathione S-transferases (GSTs) may help defend normal hepatocytes against a variety of potentially promutagenic stresses, including reactive oxygen species associated with chronic hepatic inflammation, and reactive electrophilic compounds associated with the hepatic metabolism of dietary carcinogens. Therapeutic elevation of the expression of GSTs and other carcinogen detoxification enzymes has been demonstrated to attenuate hepatic carcinogenesis in animal models. Oltipraz, an inducer of carcinogen detoxification enzyme expression in hepatocytes, is currently under development as a chemoprotective agent for human HCC.
In higher order eukaryotes DNA is methylated only at cytosines located 5xe2x80x2 to guanosine in the CpG dinucleotide. This modification has important regulatory effects on gene expression, especially when involving CpG rich areas, known as xe2x80x9cCpG islands,xe2x80x9d located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosome of females. Abberant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells, and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers.
Somatic xe2x80x9cCpG islandxe2x80x9d DNA hypermethylation changes have been frequently detected in human cancer cell genomes. Several tumor suppressor genes, such as Rb, VHL, and p16, have been reported to be inactivated by xe2x80x9cCpG islandxe2x80x9d DNA hypermethylation in different human cancer types. For HCC, changes in DNA methylation at a number of gene loci have been found to frequently accompany carcinogenesis. In one study, somatic xe2x80x9cCpG islandxe2x80x9d hypermethylation affecting E-cadherin was detected in the majority (67%) of human HCC specimens and in many (46%) liver tissues showing chronic hepatitis or cirrhosis. In another study, abnormal DNA methylation changes at several loci along chromosome 16, a chromosome frequently exhibiting allelic losses in HCC, were also detected in HCC DNA and DNA from liver tissues with chronic hepatitis or cirrhosis.
Human cancer cells typically contain somatically altered genomes, characterized by mutation, amplification, or deletion of critical genes. In addition, the DNA template from human cancer cells often displays somatic changes in DNA methylation. However, the precise role of abnormal DNA methylation in human tumorigenesis has not been established. DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA Several biological functions have been attributed to the methylated bases in DNA The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has, so far, been observed only in bacteria. Mammalian cells, however, possess a different methylase that exclusively methylates cytosine residues on the DNA, that are 5xe2x80x2 neighbors of guanine (CpG). This methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes.
A CpG rich region, or xe2x80x9cCpG islandxe2x80x9d, has recently been identified at 17p13.3, which is aberrantly hypermethylated in multiple common types of human cancers. This hypermethylation coincides with timing and frequency of 17p losses and p53 mutations in brain, colon, and renal cancers. Silenced gene transcription associated with hypermethylation of the normally unmethylated promoter region CpG islands has been implicated as an alternative mechanism to mutations of coding regions for inactivation of tumor suppressor genes. This change has now been associated with the loss of expression of VHL, a renal cancer tumor suppressor gene on 3p, the estrogen receptor gene on 6q and the H19 gene on 11p.
In eukaryotic cells, methylation of cytosine residues that are immediately 5xe2x80x2 to a guanosine, occurs predominantly in CG poor regions. In contrast, discrete regions of CG dinucleotides called CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation and parental specific imprinting where methylation of 5xe2x80x2 regulatory regions can lead to transcriptional repression. De novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas, and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas. Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated 5xe2x80x2 CpG island.
Identification of the earliest genetic changes in tumorigenesis is a major focus in molecular cancer research. Diagnostic approaches based on identification of these changes are likely to allow implementation of early detection strategies and novel therapeutic approaches targeting these early changes might lead to more effective cancer treatment.
The present invention provides for the first time that ability to detect and treat hepatic cell proliferative disorders by detecting a methylated CpG-containing glutathione-S-transferase.
In one embodiment, the invention provides a method for detecting a hepatic cell proliferative disorder by detecting a methylated CpG-containing glutathione-S-transferase (GST) nucleic acid in a hepatic specimen or biological fluid wherein a methylated GST nucleic acid is indicative a hepatic cell proliferative disorder. The method of detecting may include contacting a nucleic acid-containing hepatic specimen or biological fluid with an agent that modifies unmethylated cytosine, amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and detecting the methylated nucleic acid based on the presence or absence of amplification products produced in during amplification. Alternatively, the detection may be performed by contacting a target nucleic acid in the hepatic specimen or biological fluid with a reagent which detects methylation of the promoter region of the GST when the target nucleic acid is DNA, or wherein the reagent detects the level of GST RNA when the target nucleic acid is RNA, and detecting the GST target nucleic acid, wherein hypermethylation of the promoter of GST DNA, or decreased levels of GST RNA, as compared with the level of GST RNA in a normal cell, is indicative of a GST-associated cell proliferative disorder in hepatic tissue. The GST can be a n family GST (e.g., GSTP1).
In another embodiment, the invention provides a method for detecting a hepatic cell proliferative disorder associated with a glutathione-S-transferase (GST) in a subject by contacting a target nucleic acid in a sample of hepatic tissue or biological fluid from the subject with a reagent which detects the GST, wherein the reagent detects methylation of the promoter region of the GST when the target nucleic acid is DNA, and wherein the reagent detects the level of GST RNA when the target nucleic acid is RNA, and detecting the GST target nucleic acid, wherein hypermethylation of the promoter of GST DNA, or decreased levels of GST RNA, as compared with the level of GST RNA in a normal cell, is indicative of a GST-associated cell proliferative disorder in hepatic tissue.
In yet another embodiment, the inventin provides a method for detecting a hepatic cell proliferative disorder associated with a glutathione-S-transferase (GST) nucleic acid in a subject. The method includes contacting a target cellular component containing a GST nucleic acid with a reagent which reacts with the GST nucleic acid and detecting hypermethylation of the GST nucleic acid, wherein hypermethylation of the GST nucleic acid is indicative of a hepatic cell proliferative disorder.
In another embodiment, the invention provides a method for detecting a hepatic cell proliferative disorder associated with a glutathione-S-transferase (GST) in a subject. The method includes contacting a sample from the subject with a reagent that detects GST polypeptide and comparing the level of GST polypeptide in the sample to a control sample wherein a reduced level in the sample is indicative of a hepatic cell proliferative disorder.
In yet another embodiment, the invention provides a method for treating a hepatic cell proliferative disorder. The method includes contacting a subject in need of such treatment with an agent which increases the expression of a glutathione-S-transferase (GST), thereby treating the hepatic cell proliferative disorder.
In another embodiment, the invention provides a kit useful for the detection of a methylated CpG-containing nucleic acid in a GSTP1 promoter. The kit includes carrier means containing one or more containers having a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of the CpG-containing nucleic acid, wherein the primers distinguish between modified methylated and nonmethylated nucleic acid.
In yet another embodiment, the invention provides isolated oligonucteotide primer(s) for detection of a methylated CpG-containing nucleic acid wherein the primer hybridizes with a target polynucleotide sequence having the sequence in the region from about xe2x88x92539 to xe2x88x92239 upstream from GSTP1 transcription start site.